Hemoglobinopathies encompass a number of anemias of genetic origin in which there is decreased production and/or increased destruction (hemolysis) of red blood cells (RBCs). The blood of normal adult humans contains hemoglobin (designated as HbA) which contains two pairs of polypeptide chains designated alpha and beta. Fetal hemoglobin (HbF), which produces normal RBCs, is present at birth, but the proportion of HbF decreases during the first months of life and the blood of a normal adult contains only about 2% HbF. There are genetic defects which result in the production by the body of abnormal hemoglobins with a concomitant impaired ability to maintain oxygen concentration. Among these genetically derived anemias are included thalassemia, Cooley's Disease and sickle cell disease.
Sickle cell disease (SCD) is one of the most prevalent autosomal recessive diseases worldwide. SCD became the first genetic disorder for which a causative mutation was identified at the molecular level: the substitution of valine for glutamic acid in human βA-globin codon 6 (Ingram (1957) Nature, 180:326). In homozygotes the abnormal hemoglobin (Hb) [HbS (α2βS2)] polymerizes in long fibers upon deoxygenation within red blood cells (RBCs), which become deformed or “sickled,” rigid, and adhesive, thereby triggering microcirculation occlusion, anemia, infarction, and organ damage (Stamatoyannopoulos, et al. (eds) (1994) The Molecular Basis of Blood Diseases, Saunders, Philadelphia, ed. 2; 207-256; Nagel, et al. (2001) Disorders of Hemoglobin, Cambridge Univ. Press, Cambridge; 711-756).
Human γ-globin is a strong inhibitor of HbS polymerization, in contrast to human βA-globin, which is effective only at very high concentrations (Bookchin et al. (1971) J. Mol. Biol. 60:263). Hence, gene therapy of SCD was proposed by means of forced expression of γ-globin or γ/β hybrids in adult RBCs after gene transfer to hematopoietic stem cells (HSCs) (McCune et al. (1994) PNAS USA 91:9852; Takekoshi et al. (1995) PNAS USA 92:3014; Miller et al. (1994) PNAS USA 91:10183; Emery et al. (1999) Hum. Gene Ther. 10:877; Rubin et al. (2000) Blood 95: 3242; Sabatino et al. (2000) PNAS USA 97:13294; Blouin et al. (2000) Nat. Med. 6:177).
Although the discovery of the human β-globin locus control region (LCR) held promise to achieve high globin gene expression levels (Tuan et al. (1985) PNAS USA 82:6384; Grosveld et al. (1987) Cell 51:975), the stable transfer of murine onco-retroviral vectors encompassing minimal core elements of the LCR proved especially challenging (Gelinas et al. (1992) Bone Marrow Transplant 9:157; Chang et al. (1992) PNAS USA 89:3107; Plavec et al. (1993) Blood 81:1384; Leboulch et al. (1994) EMBO J 13:3065; Sadelain et al. (1995) PNAS USA 92:6728; Raftopoulos et al. (1997) Blood 90:3414; Kalberer et al. (2000) PNAS USA 97:5411). To allow the transfer of larger LCR and globin gene sequences, one proposal was the use of RNA splicing and export controlling elements that include the Rev/R responsive element (RRE) components of human immunodeficiency virus (HIV) (Alkan et al. (31 May 2000) paper presented at the 3rd American Society of Gene Therapy, Denver, Colo.), and an RRE-bearing HIV-based lentiviral vector which had resulted in substantial amelioration of 13-thalassemia in transplanted mice (May et al. (2000) Nature 406:82). This approach was not sufficient for complete correction, however, as gene expression remained heterocellular, and the amount of human βA-globin found incorporated in Hb tetramers in a nonthalassemic background was unlikely to be successful therapy for SCD (May et al., supra). Accordingly, there remains a need for a gene therapy approach which can successfully treat SCD and other hemoglobinopathies.